Trichoderma reesei engineering bacteria for high yield of sorbicillinoids and construction method and application of trichoderma reesei engineering bacteria

A technology of Trichoderma reesei and a construction method, which is applied in the field of bioengineering, can solve the problems that the research has not been reported, the influence of the synthesis of Sorbicillinoids is not very clear, etc., and achieves the effect of increasing yield and stabilizing hereditary traits.

Active Publication Date: 2021-03-26
SHANXI MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the effect of H3K9 methylation modification on the synthesis of Sorbicillinoids in Trichoderma reesei is not very clear, and its research on the synthesis mechanism of Sorbicillinoids in Trichoderma reesei has not been reported

Method used

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  • Trichoderma reesei engineering bacteria for high yield of sorbicillinoids and construction method and application of trichoderma reesei engineering bacteria
  • Trichoderma reesei engineering bacteria for high yield of sorbicillinoids and construction method and application of trichoderma reesei engineering bacteria
  • Trichoderma reesei engineering bacteria for high yield of sorbicillinoids and construction method and application of trichoderma reesei engineering bacteria

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Experimental program
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Effect test

Embodiment 1

[0051] Embodiment 1: Construction of Trichoderma reesei ΔTrclr4 engineering bacteria

[0052] 1. Construction of pDonor-pyr4-ΔTrclr4 gene knockout vector

[0053] 1) According to the genomic DNA sequence of Trichoderma reesei, the amplification primers for the upstream and downstream homology arms of the Trclr4 gene (SEQ ID NO.1) were designed, and the amplification primers for the upstream homology arms were clr4-up-F and clr4-up-R, The downstream homology arm amplification primers are clr4-down-F and clr4-down-R; wherein, the primer sequences of the clr4-up-F, clr4-up-R, clr4-down-F and clr4-down-R as follows:

[0054] clr4-up-F: 5′-TAGGGATAACAGGGTAATCGATGGCCCAGCGTAGAA-3′,

[0055] clr4-up-R: 5′-GGGGACAAGTTTGTACAAAAAAAGCAGGCTAAATAACCTCCTGAATCAGTCGTA-3′,

[0056] clr4-down-F: 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTACAAACCGCATCATCCAAC-3′,

[0057] clr4-down-R: 5′-ATTACCCTGTTATCCCCTATCATGGCTGGCCTGATTT-3′;

[0058] 2) Extract the genomic DNA of Trichoderma reesei QM9414, take the ...

Embodiment 2

[0086] Example 2: Analysis of transcription level of main genes involved in sorbicillinoids synthesis and sorbicillinoids secretion level analysis in Trichoderma reesei ΔTrclr4 engineering bacteria

[0087] 1. Use the qRT-PCR method to analyze the gene transcription levels of ypr1, ypr2, sor1, sor2, sor3, sor4 and sor6 in Trichoderma reesei ΔTrclr4 engineering bacteria, the steps are as follows:

[0088] 1) The control strain QM9414 and the constructed Trichoderma reesei ΔTrclr4 engineering bacteria were inoculated on MA medium with 1% (v / v) glycerol as carbon source for pre-cultivation, at 30° C., 200 rpm for 48 h; collected mycelia, Then transfer to a fermentation medium with 1% (w / v, g / mL) microcrystalline cellulose as a carbon source, and continue to cultivate at 30°C and 200rpm;

[0089] Wherein, the MA medium components are as follows: 17.907g / L Na 2 HPO 4 12H 2 O,2g / L KH 2 PO 4 ,1.4g / L(NH4) 2 SO 4 , 0.3g / L urea, 0.5ml / L Tween-80, adjust pH to 5.0 with anhydrous c...

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Abstract

The invention relates to trichoderma reesei engineering bacteria for high yield of sorbicillinoids and a construction method and application of the trichoderma reesei engineering bacteria. The trichoderma reesei engineering bacteria disclosed by the invention are constructed by knocking out genes Trclr4 of trichoderma reesei with trichoderma reesei QM9414 as an original strain. By knocking out thecoding gene Trclr4 of histone H3K9 methylase of the trichoderma reesei, the important role of histone H3K9 methylation in synthesis of sorbicillinoids from the trichoderma reesei is proved for the first time, and high yield of the sorbicillinoids in the trichoderma reesei is realized. Compared with the original strain, the trichoderma reesei delta Trclr4 engineering bacteria have the advantage that the yield of the sorbicillinoids under the condition of taking cellulose as a carbon source is doubled.

Description

technical field [0001] The invention relates to a high-yield sorbicillinoids Trichoderma reesei engineering bacterium and its construction method and application, belonging to the technical field of bioengineering. Background technique [0002] Secondary metabolites are not necessary for cell growth, but they often have high biological activity and have potential application value in the fields of biomedicine and human health industry. Among them, Sorbicillinoids (also commonly referred to as yellow pigments) are a class of fungal secondary metabolites synthesized by polyketide synthase and have antitumor, antiviral, antioxidative, and antibacterial biological activities. So far, more than 90 species of Sorbicillinoids have been discovered, which can be synthesized and secreted by fungi from land and sea sources. Based on its structure, it can be mainly divided into 4 categories, monomeric sorbicillinoids, binary bisorbicillinoids, ternary trisorbicillinoids and hybrid sorb...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12N15/80C12N15/65C12P7/26C12R1/885
CPCC12N15/80C12N15/65C12N9/1007C12P7/26
Inventor 王磊梁宇翔解军
Owner SHANXI MEDICAL UNIV
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